Experimental study of plantamajoside in myocardial injury mice with sepsis induced by lipopolysaccharide
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摘要:目的
探讨大车前苷在小鼠脓毒症心肌损伤中的作用。
方法选取8~10周龄的雄性C57/BL6小鼠40只, 根据处理方式不同将小鼠随机分为4组: 生理盐水+生理盐水组、生理盐水+大车前苷组、脂多糖+生理盐水组、脂多糖+大车前苷组，每组10只。脂多糖+生理盐水组与脂多糖+大车前苷组小鼠接受单次腹腔注射脂多糖(10 mg/kg), 以构建小鼠脓毒症模型; 生理盐水+生理盐水组与生理盐水+大车前苷组小鼠接受同等体积生理盐水腹腔注射。生理盐水+大车前苷组与脂多糖+大车前苷组小鼠给予连续5 d的大车前苷50 mg/(kg·d)灌胃干预，生理盐水+生理盐水组与脂多糖+生理盐水组进行同等体积生理盐水灌胃。实验第1天，先给予小鼠连续5 d大车前苷50 mg/(kg·d)或生理盐水灌胃，第5天给予小鼠单次腹腔注射脂多糖(10 mg/kg)或者等体积生理盐水，饲养12 h后检测心功能并取材。采用实时荧光定量聚合酶链反应检测超氧化物歧化酶2 (SOD-2)、谷胱甘肽过氧化物酶-1 (GPX-1)和过氧化氢酶(CAT)和相关炎症因子[白细胞介素-1β(IL-1β)、白细胞介素-6 (IL-6)、肿瘤坏死因子-α (TNF-α)、单核细胞趋化蛋白-1 (MCP-1)和白细胞介素-4 (IL-4)]的mRNA水平。用检测试剂盒检测丙二醛(MDA)、4-羟基壬烯醛(4-HNE)、GPX-1、TNF-α和MCP-1以及Caspase-3的水平; 检测血液中心肌肌钙蛋白I(cTnI)、乳酸脱氢酶(LDH)水平; 用TUNEL染色检测心肌细胞凋亡水平。
结果与生理盐水+生理盐水组小鼠相比，脂多糖+生理盐水组小鼠的心率、左室射血分数以及左室短轴缩短率降低，心肌损伤标志物cTnI、LDH水平升高，差异有统计学意义(P < 0.05); 大车前苷可恢复小鼠的心率、左室射血分数、左室短轴缩短率，以及降低心肌损伤标志物cTnI和LDH的水平，提高小鼠生存率(P < 0.05)。与脂多糖+生理盐水组小鼠相比，脂多糖+大车前苷组小鼠心脏中MDA、4-HNE的水平降低，差异有统计学意义(P < 0.05)。大车前苷可降低小鼠心脏中炎症因子表达、Caspase-3的活性、细胞凋亡水平(P < 0.05)。
结论大车前苷可以减轻脂多糖诱导的小鼠心肌细胞损伤，改善其心功能。
Abstract:ObjectiveTo investigate the role of plantamajoside in sepsis-related cardiac injury in mice.
MethodsForty male C57/BL6 mice aged 8 to 10 weeks were selected and randomly divided into 4 groups according to different treatment methods: normal saline+normal saline group, normal saline+plantamajoside group, lipopolysaccharide+normal saline group, lipopolysaccharide+plantamajoside group, with 10 mice in each group. Mice in lipopolysaccharide+normal saline group and lipopolysaccharide+plantamajoside group received single intraperitoneal injection of lipopolysaccharide (10 mg/kg) to construct a mouse sepsis model; mice in the normal saline+normal saline group and the normal saline+plantamajoside group received intraperitoneal injection of the same volume of normal saline. The mice in the normal saline+plantamajoside group and the lipopolysaccharide+plantamajoside group were given 50 mg/(kg·d) plantamajoside by gavage intervention for consecutive 5 days, and the mice in the normal saline+normal saline group and the lipopolysaccharide+normal saline group were given gavage with the same volume of normal saline. On the first day of the experiment, the mice were given 50 mg/(kg·d) or normal saline intragastric administration for 5 consecutive days. On the fifth day, mice were given a single intraperitoneal injection of lipopolysaccharide (10 mg/kg) or equal volume of normal saline. After feeding for 12 h, cardiac function was detected and samples were collected. Superoxide dismutase 2 (SOD-2), glutathione peroxidase-1 (GPX-1), catalase (CAT) and related inflammatory factors [interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and interleukin-4 (IL-4)] mRNA levels were determined by real-time quantitative fluorescence polymerase chain reaction. The levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), GPX-1, TNF-α, MCP-1 and Caspase-3 were determined by the test kit; the serum levels of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were detected; the myocardial cell apoptosis was detected by TUNEL staining.
ResultsCompared with normal saline + normal saline group, the heart rate, left ventricular ejection fraction and left ventricular short axis shortening rate of mice in lipopolysaccharide + normal saline group were significantly decreased, and the myocardial injury markers including cTnI and LDH were significantly increased (P < 0.05). Plantamajoside could restore the heart rate, left ventricular ejection fraction, left ventricular short axis shortening rate, reduce the levels of myocardial injury markers including cTnI and LDH, and improve the survival rate of mice (P < 0.05). Compared with the lipopolysaccharide+normal saline group, the levels of MDA and 4-HNE in the heart of mice in lipopolysaccharide+plantamajoside group were significantly decreased (P < 0.05). Plantamajoside could decrease the expression of inflammatory factors, the activity of Caspase-3 and the level of apoptosis in the heart of mice (P < 0.05).
ConclusionPlantamajoside can alleviate myocardial cell damage induced by lipopolysaccharide and improve cardiac function in mice.
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Keywords:
- plantamajoside /
- sepsis /
- myocardial injury /
- inflammation /
- oxidative stress
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图 1 各组小鼠心功能比较
A: 小鼠心率; B: 射血分数; C: 左室短轴缩短率; D: 生存率。与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
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图 2 各组小鼠心肌损伤标志物
A: cTnI水平; B: LDH水平。与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
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图 3 各组小鼠心脏MDA、4-HNE相对水平
A: MDA水平; B: 4-HNE水平。与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
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图 4 各组小鼠心脏中SOD-2、GPX-1以及CAT的mRNA相对水平
与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
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图 5 各组小鼠心脏中TNF-α以及MCP-1相对水平
A: TNF-α相对水平; B: MCP-1相对水平。与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
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图 6 各组小鼠心脏中IL-1、IL-6、TNF-α、MCP-1以及IL-4的mRNA相对水平
与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
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图 7 各组小鼠心脏中Caspase-3活性以及细胞凋亡情况
A: Caspase-3活性相对水平; B: TUNEL阳性率; C: 细胞凋亡情况。与生理盐水+生理盐水组比较, *P < 0.05; 与脂多糖+生理盐水组比较, #P < 0.05。
表 1 相关指标引物序列
引物名称 上游引物5′- 3′ 下游引物5′- 3′ SOD-2 CAGACCTGCCTTACGACTATGG CTCGGTGGCGTTGAGATTGTT GPX-1 AGTCCACCGTGTATGCCTTCT GAGACGCGACATTCTCAATGA CAT AGCGACCAGATGAAGCAGTG TCCGCTCTCTGTCAAAGTGTG IL -1β GCAACTGTTCCTGAACTCAACT ATCTTTTGGGGTCCGTCAACT IL -6 TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC TNF-α CCCTCACACTCAGATCATCTTCT GCTACGACGTGGGCTACAG MCP-1 TTAAAAACCTGGATCGGAACCAA GCATTAGCTTCAGATTTACGGGT IL-4 GGTCTCAACCCCCAGCTAGT GCCGATGATCTCTCTCAAGTGAT GAPDH AGGTCGGTGTGAACGGATTTG AGGTCGGTGTGAACGGATTTG 
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